Subsequent Bioassay Research Articles

Centipeda cunninghamii, an australian traditional medicinal plant

Centipeda cunninghamii (DC.) A.Braun & Asch. is an endemic Australian Asteraceae with a long history of traditional use as a medicinal plant for treating wounds, infections and inflammation. Whilst its essential oil composition, principally chrysanthenyl and sabinyl acetates, has been known for some time, there was little scientific information regarding its phytochemistry and biological activity. Investigations on aqueous ethanolic extracts confirmed its anti-inflammatory and antioxidant (ORAC) activity. Detailed investigations suggest the extract acts against a range of inflammatory markers including COX-1, COX-2, NO and TNF-α, but not through the lipoxygenase pathway. Seventeen compounds were isolated and subsequent bioassays indicated that the anti-inflammatory activity was linked to flavonoids, whilst the antioxidant activity was attributed to both flavonoids and a group of novel heptenedioic acid cinnamoyl esters. The latter compounds are ring-opened quinic acid derivatives and appear to be unique to this species. Optimisation of growing, post-harvest and extraction conditions based on quality markers have been developed for future production and product development.

Exploration of acridine scaffold as a potentially interesting scaffold for discovering novel multi-target VEGFR-2 and Src kinase inhibitors

VEGFR-2 and Src kinases both play important roles in cancers. In certain cancers, Src works synergistically with VEGFR-2 to promote its activation. Development of multi-target drugs against VEGFR-2 and Src is of therapeutic advantage against these cancers. By using molecular docking and SVM virtual screening methods and based on subsequent synthesis and bioassay studies, we identified 9-aminoacridine derivatives with an acridine scaffold as potentially interesting novel dual VEGFR-2 and Src inhibitors. The acridine scaffold has been historically used for deriving topoisomerase inhibitors, but has not been found in existing VEGFR-2 inhibitors and Src inhibitors. A series of 21 acridine derivatives were synthesized and evaluated for their antiproliferative activities against K562, HepG-2, and MCF-7 cells. Some of these compounds showed better activities against K562 cells in vitro than imatinib. The structure–activity relationships (SAR) of these compounds were analyzed. One of the compounds ( 7r) showed low μM activity against K562 and HepG-2 cancer cell-lines, and inhibited VEGFR-2 and Src at inhibition rates of 44% and 8% at 50 μM, respectively, without inhibition of topoisomerase. Moreover, 10 μM compound 7r could reduce the levels of activated ERK1/2 in a time dependant manner, a downstream effector of both VEGFR-2 and Src. Our study suggested that acridine scaffold is a potentially interesting scaffold for developing novel multi-target kinase inhibitors such as VEGFR-2 and Src dual inhibitors.

Isolation of quinone reductase (QR) inducing agents from ginger rhizome and their in vitro anti-inflammatory activity

To investigate the potential activity of ginger rhizome extracts to induce quinone reductase (QR), we performed bioactivity-guided fractionation using a murine hepatoma cell (Hepa 1c1c7) bioassay. Anti-inflammatory effects were then studied utilizing lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW 264.7) cells. An ethyl acetate-partitioned fraction from ethanolic extract, rich in both QR inducing potency and anti-inflammatory activity, was subjected to repeated silica gel column and preparative thin layer chromatography to yield three compounds. The three isolated compounds were [6]-shogaol, 1-dehydro-[6]-gingerdione and hexahydrocurcumin. [6]-Dehydroshogaol, a minor component in ginger rhizome, was chemically synthesized and used for comparison in the subsequent bioassay based on its excellent QR inducing potency. Results showed that [6]-dehydroshogaol had the highest ability to induce QR activity (CD = 9.23 ± 0.22 μM), followed by 1-dehydro-[6]-gingerdione (CD = 13.24 ± 0.45 μM), and then hexahydrocurcumin (CD = 68.81 ± 3.90 μM). Increasing QR activity in induced cells was associated with elevated expression of NQO-1 protein as confirmed by Western blot. [6]-Dehydroshogaol, [6]-shogaol and 1-dehydro-[6]-gingerdione were also potent inhibitors of nitric oxide (NO) synthesis in activated macrophages. Their IC 50 values ranged from 5.80 ± 1.27 to 25.06 ± 4.86 μM. Hexahydrocurcumin exhibited the weakest inhibitory effect (IC 50 = 304.76 ± 54.80 μM). These findings contribute to our theoretical understanding of the potential beneficial effects of consuming ginger as food and/or dietary supplement.

Phylogenetic diversity of Gram-positive bacteria cultured from Antarctic deep-sea sponges

Gram-positive bacteria, specifically actinobacteria and members of the order Bacillales, are well-known producers of important secondary metabolites. Little is known about the diversity of Gram-positive bacteria associated with Antarctic deep-sea sponges. In this study, cultivation-based approaches were applied to investigate the Gram-positive bacteria associated with the Antarctic sponges Rossella nuda, Rossella racovitzae (Porifera: Hexactinellida), and Myxilla mollis, Homaxinella balfourensis, Radiella antarctica (Porifera: Demospongiae). In total, 46 Gram-positive strains were cultured. Phylogenetic analysis revealed that 24 strains were affiliated with the Actinobacteria, including six genera Streptomyces, Nocardiopsis, Pseudonocardia, Dietzia, Brachybacterium, and Brevibacterium. The other 22 strains were affiliated with the Firmicutes, and among them two (V17-1 and V179-1) only shared 92–95% 16S rRNA gene sequence identity with the nearest type strain. To our knowledge, this is the first report on the isolation of strains belonging to genera Dietzia and Brevibacterium from Antarctic sponges. All of the 46 strains were PCR screened for genes encoding polyketide synthases (PKS), and a selection of 36 isolates were used in subsequent bioassay analyses. Eighty-eight percentage of the isolates that possess a PKS gene were active against at least one test organism. The study confirms the existence of diverse bacteria in Antarctic sponges and their potential for producing active compounds.

Dose-response testing of Australian populations of onion thrips Thrips tabaci Lindeman (Thysanoptera: Thripidae) further refines baseline data and detects methidathion and likely imidacloprid resistance

Field strains of Thrips tabaci Lindeman were collected from bulb onion in New South Wales, Queensland and South Australia and returned to the laboratory for culturing and subsequent bioassay to determine chemical specific responses. Chemicals tested included diazinon, dimethoate, λ-cyhalothrin and methidathion with imidacloprid and spinetoram evaluated to establish reliable susceptible baseline responses. Reference (susceptible) responses were compared back with those previously published and for all insecticides more susceptible strains were found for resistance comparison. Results for methidathion in particular were found to be significantly different from those reported previously. These differences influenced resistance factor calculation considerably. A high 49-fold methidathion resistance was detected, a result consistent with anecdotal grower concerns of poor product performance against T. tabaci. Resistance to diazinon remains generally low with all strains tested showing <6-fold at the LC50 level. LC50 dimethoate resistance peaked at 17-fold. Maximum (56-fold) λ-cyhalothrin resistance was significantly less than previously found but responses were very heterogeneous implying resistance could very quickly increase. Worryingly, the difference in imidacloprid responses between the least and most tolerant was 41-fold although the chemical is neither used nor registered for use against T. tabaci. We consider such a high and significant difference in response to imidacloprid is most likely caused by an unknown cross-resistance that may compromise any future development of imidacloprid for use against T. tabaci. Thrips remain susceptible to spinetoram.

Chronic Progressive Nephropathy in Male F344 Rats in 90-Day Toxicity Studies

The occurrence and severity of spontaneous chronic progressive nephropathy (CPN) in control male F344 rats as well as the frequency of treatment-related CPN exacerbation were histopathologically reevaluated. A series of 43 National Toxicology Program (NTP) 90-day toxicity studies comparing the influence of NIH-07 or NTP-2000 diets was examined. Relationships between the histopathologic findings at 90 days and renal tubule proliferative lesions recorded in subsequent 2-year bioassays for 24 chemicals were statistically analyzed. CPN lesions were observed in 100% of the control male rats regardless of diet, but CPN was more severe in control rats fed NIH-07. Approximately one-third of the 90-day studies demonstrated a treatment-related exacerbation of CPN severity, which was independent of diet. For chemicals that proceeded to 2-year bioassays, all studies with a statistically significant increase in renal tubule tumors (RTT) at 2 years had treatment-related exacerbation of CPN in the 90-day and 2-year studies. These findings indicate that CPN occurs ubiquitously in young male F344 rats and that treatment-related exacerbation of CPN in 90-day studies is a relatively common occurrence, having the potential to be predictive of an increased incidence of RTT in subsequent 2-year bioassays.

The Main Virulence Determinant of Yersinia entomophaga MH96 Is a Broad-Host-Range Toxin Complex Active against Insects

Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ~32-kb pathogenicity island (PAI) designated PAI(Ye₉₆). Residing within PAI(Ye₉₆) are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (~19 kb) of PAI(Ye₉₆) was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ΔTC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ΔTC mutant strain could be restored by the introduction of a clone containing the entire PAI(Ye₉₆) TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ΔTC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD₅₀s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

Objectives:The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide.Materials and Methods:Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay.Results:A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical –KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation.Conclusion:IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Kin Recognition in the largely Solitary Bee,Manuelia postica(Apidae: Xylocopinae)

The recognition of conspecifics is a central issue to social behaviour. In eusocial hymenopterans, kin recognition has been clearly demonstrated. Manuelia postica is a largely solitary bee species in which larvae develop inside individual cells within a nest and remain isolated from conspecifics until the destruction of partitions by adults. Nestmate recognition in M. postica has been previously demonstrated under experimental conditions. Isolation between individuals during development and nestmate recognition ability in adult females make M. postica an ideal species for testing the occurrence of kin recognition capacity in females. Kin recognition was demonstrated through cross-fostering field experiments involving the single transfer of recently enclosed larvae, and subsequent laboratory recognition bioassays with emerging females. Results suggest kin recognition occurs through self-referent phenotype matching. Given the basal position of Manuelia in the phylogeny of the Apidae, kin recognition may represent an ancestral recognition mechanism in Apidae species phylogenetically more derived than M. postica.

Expanding the application scope of glycosidases using click chemistry

Glycosidase-mediated glycosylation of alkynyl alcohols and azide-containing alcohols was followed by a click reaction, affording various types of triazole glycosides. The activities of triazole glycosides detected in subsequent bioassays show that this procedure is a feasible approach to the development of anti-fungal drugs.

Recognition of Host Plant Volatiles by Pheidole minutula Mayr (Myrmicinae), an Amazonian Ant‐Plant Specialist

ABSTRACTIn the tropics, several ant species are obligate inhabitants of leaf pouches and other specialized structures in plants known as myrmecophytes. However, the cues used by ant queens to locate suitable host‐plants following dispersal remain poorly understood. Here we tested the hypothesis that Pheidole minutula queens use volatiles to distinguish their host Maieta guianensis (Melastomataceae) from other sympatric myrmecophytes. To do so, we used a Y‐tube olfactometer to quantify the preference for volatiles of different plant species. Our results indicate that P. minutula queens discriminate the chemical volatiles produced by its host‐plant from those of other sympatric ant‐plant species. However, queens failed to distinguish the volatiles of Maieta from those of the ant‐plant Tococa bullifera (Melastomataceae), with which P. minutula is not mutualistically associated. Nevertheless, a strong preference for Maieta over Tococa was observed during a subsequent bioassay, where the ants had physical contact with a domatium of each plant species. These results suggest that additional, short distance mechanisms are also necessary for host discrimination. Overall, our findings suggest that the high degree of compartmentalization observed in symbiotic ant–plant relationships is achieved, at least in part, by the relatively high degree of specificity in host selection displayed by foundress queens.

Chemical Signalling: Laser on the Fly Reveals a New Male-Specific Pheromone

A study using laser desorption/ionization mass spectrometry has identified new oxygenated compounds in cuticular lipids of the fruit fly Drosophila melanogaster; one such compound, when transferred to females during mating, renders the females unattractive to males for days.

Separation and toxicity of salithion enantiomers

Enantioseletive toxicities of chiral pesticides have become an environmental concern recently. In this study, we evaluated the enantiomeric separation of salithion on a suite of commercial chiral columns and assessed the toxicity of enantiomers toward butyrylcholinesterase and Daphnia magna. Satisfactory separations of salithion enantiomers could be achieved on all tested columns, that is, Chiralcel OD, Chiralcel OJ, and Chiralpak AD column. However, the Chiralpak AD column offered the best separation and was chosen to prepare micro-scale of pure salithion enantiomers for subsequent bioassays. The first and second enantiomers eluted on the Chiralpak AD column were further confirmed to be (-)-S-salithion and (+)-R-salithion, respectively. The half inhibition concentrations to butyrylcholinesterase of racemate, (+)-R-salithion, and (-)-S-salithion were 33.09, 2.92, and 15.60 mg/l, respectively, showing (+)-R-enantiomer being about 5.0 times more potent than its (-)-S-form. However, the median lethal concentrations (96 h) of racemate, (+)-R-salithion, and (-)-S-salithion toward D. magna were 3.54, 1.10, and 0.36 microg/l, respectively, suggesting that (-)-S-salithion was about 3.0 times more toxic than (+)-R-form. Racemic salithion was less toxic than either of the enantiomers in both bioassays, suggesting that antagonistic interactions might occur between the enantiomers during the toxication action. This work reveals that the toxicity of salithion toward butyrylcholinesterase and D. magna is enantioselective, and this factor should be taken into consideration in the environmental risk assessment of salithion.

Light-activated cytotoxic compounds from Malaysian microorganisms for photodynamic therapy of cancer

Photodynamic therapy (PDT) is a promising cancer treatment which involves activation of a photosensitizing drug with light to produce reactive oxygen species that kill tumors without causing damage to unirradiated normal tissues. To date, only Photofrin, Foscan and Levulan have been approved for clinical treatment of cancer. Tropical habitats such as those found in Malaysia are attractive sources of new therapeutic compounds as tremendous chemical diversity is found in a large number of plants, animals, marine- and micro-organisms. In our screening program for novel photosensitizers from nature, colorful strains of fungi (from Aspergillus and Penicillium genus) and bacteria (including actinomycetes and photosynthetic bacteria) were collected from various habitats in Peninsular Malaysia, such as coastal soil, peat soil, marine sponges and wastewater ponds. Methanolic extracts from a total of 85 different species were evaluated with a short-term cell viability assay for photo-cytotoxicity, where a promyelocytic leukemia cell-line, HL60 incubated with 20 microg/ml of extracts was irradiated with 9.6 J/cm(2) of a broad spectrum light. Two of these extracts, one from Rhodobacter sphaeroides (PBUM003) and one from Rhodopseudomonas palustris (PBUM001) showed moderate to strong photo-cytotoxicity. Subsequent bioassay guided isolation of the PBUM001 extract yielded known photosensitisers that are based on bacteriochlorophyll-a by comparing their molecular weight data, HPLC profiles and UV-vis absorption spectra with literature values, thereby demonstrating the validity of our screening approach.

Oxidation of lanthionines renders the lantibiotic nisin inactive.

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the beta-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.

Screening of arbuscular mycorrhizal fungal consortia developed from the rhizospheres of natural and cultivated tea plants for growth promotion in tea [ Camellia sinensis (L.) O. Kuntze

Summary Soil-based arbuscular mycorrhizal (AM) fungal consortia were developed from the soil of natural tea rhizosphere (NTR) and cultivated tea rhizosphere (CTR) located in the Indian Himalayan region. Plant growth promotion following inoculation with these consortia was studied. Bioassays were conducted under greenhouse conditions using maize and wheat as test plants. Both treatments (NTR and CTR) significantly increased the growth of maize and wheat plants to different degrees. A subsequent bioassay was conducted with tea [ Camellia sinensis (L.) O. Kuntze] plants. Both AM fungal consortia were found to promote growth of tea plants significantly in non-sterilized acidic soil. Data suggest that crude consortia can promote plant growth; more efficiently in case of AM fungal consortia developed from NTR compared to CTR where cultural practices can reduce the potential of native AM fungi. The NTR consortium has significant potential as a biofertilizer in tea plantations of colder regions.

Inhaled ultrafine particulate matter affects CNS inflammatory processes and may act via MAP kinase signaling pathways

In addition to evidence that inhalation of ambient particulate matter (PM) can increase cardiopulmonary morbidity and mortality, the brain may also constitute a site adversely effected by the environmental presence of airborne particulate matter. We have examined the association between exposure to PM and adverse CNS effects in apolipoprotein E knockout (ApoE −/−) mice exposed to two levels of concentrated ultrafine particulate matter in central Los Angeles. Mice were euthanized 24 h after the last exposure and brain, liver, heart, lung and spleen tissues were collected and frozen for subsequent bioassays. There was clear evidence of aberrant immune activation in the brains of exposed animals as judged by a dose-related increase in nuclear translocation of two key transcription factors, NF-κB and AP-1. These factors are involved in the promotion of inflammation. Increased levels of glial fibrillary acidic protein (GFAP) were also found consequent to particulate inhalation suggesting that glial activation was taking place. In order to determine the mechanism by which these events occurred, levels of several MAP kinases involved in activation of these transcription factors were assayed by Western blotting. There were no significant changes in the proportion of active (phosphorylated) forms of ERK-1, IkB and p38. However, the fraction of JNK in the active form was significantly increased in animals receiving the lower concentration of concentrated ambient particles (CAPs). This suggests that the signaling pathway by which these transcription factors are activated involves the activation of JNK.

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone ( Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Virulence of entomopathogenic hypocrealean fungi infecting Anoplophora glabripennis

Twenty isolates of four species of entomopathogenic hypocrealean fungi (Beauveria bassiana, Beauveria brongniartii, Isaria farinosa, and Metarhizium anisopliae) were found to be pathogenic to adults of the Asian longhorned beetle, Anoplophora glabripennis. Survival times for 50% of the beetles tested (ST50) ranged from 5.0 (M. anisopliae ARSEF 7234 and B. brongniartii ARSEF 6827) to 24.5 (I. farinosa ARSEF 8411) days. Screening studies initially included strains of B. brongniartii, which is registered as a microbial control agent in Europe, Asia and South America but not in North America. At that time, we could not confirm that this fungal species is native to North America which added uncertainty regarding future registration of this species for pest control in the USA. Therefore, subsequent bioassays documented median survival times for three M. anisopliae isolates (5-6 days to death) and two of these isolates are suggested for further development because they are already registered for pest control in the USA.

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2′ O)-methyltransferase

We report high-throughput structure-based virtual screening of putative Flavivirus 2′- O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate 7MeGpppAC 5. Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC 50 value of 60 μM.